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Liquid Chromatography
Preliminary Lab Assignment
1. What is the process of chromatography used for? (use the internet)
2. In chromatography, components of a mixture spend some time adsorbed on a stationary
phase and some time dissolved in a mobile phase. Explain how the components can be
separated with these two phases.
3. In the liquid chromatography column used in this experiment, the solid has a C18
hydrocarbon bonded to it. Would a C 18 hydrocarbon be a polar or a nonpolar substance?
Explain.
4. The Kool-Aid® that is to be separated in this experiment consists of citric acid, calcium
phosphate, salt, maltodextrin, artificial flavor, ascorbic acid, FD&C Red #40 and FD&C
Blue #1 dyes. Group these as very polar, moderately polar, or nonpolar.
5. Suggest a different mixture for which liquid chromatography might be a useful separation
tool.
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Liquid Chromatography
In this experiment we will use liquid chromatography to separate the substances that are present in grape
flavored Kool-Aid®. First, the dyes FD&C Blue #1 and Red #40 will be separated. The other components of
Kool-Aid®, the flavorings and citric acid, will be separated in a second experiment.
Chromatography is an important analytical tool that is used to separate the components of a mixture. Liquid
chromatography is one type of chromatography that is enormously useful in research and in industry. High
performance liquid chromatography (HPLC) has become an almost indispensable tool for scientists. There
are many kinds of chromatography, but all have some elements in common. First, there is a stationary
support medium which attracts the components of the mixture. This medium may be polar, attracting polar
components of the mixture, or nonpolar, attracting the nonpolar components. In liquid chromatography, this
support is a column packed with a fine, granular solid. The mixture to be separated is placed in the column
and clings to the solid. The second necessary component is a solvent which washes along the column. This
solvent has a different polarity than the solid. The components of the mixture may be more strongly attracted
to the solvent or to the stationary support, depending on their polarity. As the solvent washes through the
column, the components of the mixture spend some time adsorbed on the stationary support and some time
dissolved in the moving solvent. The substances that are more soluble in the solvent travel more quickly
through the column, and emerge early. Those substances that are more strongly attracted to the stationary
support move slowly, and emerge later.
A C18 Sep-Pak® cartridge is the column that will be used in this experiment. This column is packed with a
silica solid which has a C18 hydrocarbon bonded to it, so it is very nonpolar.
A third component of chromatography is that a means of injecting the sample into the column is required.
We will use a disposable hypodermic syringe. Fourth, a pump is needed to force the solvent through the
column. We will use a syringe or plastic squeeze bottle. Next, a detector is required to tell when the
components emerge from the column. Since we will be separating colored dyes, we can use our eyes to see
the dyes as they emerge from the column. The recording of the experiment will be done manually with pen
and a laboratory notebook.
Figure 1. Components of Mixture Moving through Liquid Chromatography Column
When a mixture is injected into the liquid chromatography column and washed through it, several processes
occur. Refer to Figure 1. The more polar components of the mixture are attracted more strongly to the
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solvent, so they will move more quickly through the column with the solvent. The less polar components will
move more slowly, as they spend more time adsorbed to the column medium. Ideally, the components should
emerge at different times. A measure of the degree of separation that is achieved is called the resolution of
the system. A second process that occurs which works against resolution is that as the band of each
component moves down the column, the band widens due to diffusion. As bands widen they overlap each
other more easily and prevent clean separation or resolution of the components.
Chemicals
Isopropanol, C3H7OH, 70% or 91%, colorless, unscented
Grape Kool-Aid®, or other grape drink, unsweetened
Equipment
Sep-Pak® C 18 cartridge (Draw in my desk)
Graduated cylinders, 10-mL and 25-mL
Beakers, 4, 50-mL or 10-niL
Beakers, 4, 100-mL
Syringe, 1 mL or 2 mL with male Luer® tip
Syringe, 10-mL with male Luer® tip,
or 50-mL or 1 00-mL dropper bottles with
plastic tips,
or 100-mL or 250-mL wash bottles
Procedure
Safety Alert
Isopropyl alcohol is flammable. Keep it away from flames.
Wear Chemical Splash Goggles and a Chemical-Resistant Apron.
Isocratic separation
In an isocratic separation, the solvent composition and flow rate are held constant throughout the experiment.
The solvent composition is chosen to be able to elute both of the dyes in the grape drink at different rates. In
an isocratic separation, the resolution, selectivity and efficiency of the separation can be calculated.
1. Prepare Kool-Aid®.
Prepare the grape Kool-Aid® as directed on the package, but omit the sugar. To prepare less than a whole
package, use 0.5 g/250 mL water.
2. Prepare the isopropanol Eluant.
Prepare 18% (v/v) isopropanol in water to be used as the mobile phase. Combine 13 mL of 70% isopropanol
with 37 mL distilled water (or 10 mL 91% isopropanol with 40 mL distilled water).
3. Pretreat the C18 Sep-Pak® cartridge.
To help eliminate remixing of closely eluting bands in the cartridge, cut off the exit tube of the cartridge (the
shorter end) at the point where it meets the body of the cartridge. Pre-wet the cartridge by pumping about 10
mL of undiluted (70% isopropanol) through the cartridge.
If you are using a syringe, fill it with 10 mL of the undiluted isopropanol. Attach the tip to the long end of
the Sep-Pak® cartridge, and pump the isopropanol through the syringe at a rate of 5—10 mL per minute.
Collect the eluted alcohol in a 10 mL graduated cylinder to monitor the flow rate. If you are using a plastic
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bottle with a pointed dropper top or a wash bottle, attach the top of the filled bottle firmly to the cartridge,
and slowly pump the isopropanol through the cartridge.
Next, wash the cartridge with 10 mL of distilled water at the same flow rate.
4. Inject the sample.
Use a small (1- or 2-mL) syringe to slowly inject 1 mL of the Kool-Aid® sample onto the column. Discard
the column effluent (the portion that washed out as you injected the sample).
5. Elute the sample.
Use a 10-mL syringe or a plastic dropper bottle to slowly elute the dyes. Fill the syringe or dropper bottle
with the 18% isopropanol eluant, and pump at a steady rate of 5—10 mL per minute. Collect the effluent in a
10-mL graduated cylinder. Record the volume of effluent collected as the first and last of the colored drops
of each of the dyes emerge. If there is not a perfect separation between the blue- and red-colored bands,
record data for the beginning and end of the intermediate purple The center of the purple band will serve as
the end of the first band and beginning of the last.
6. Regenerate the cartridge and repeat the measurements.
Repeat the measurements two more times. Show all your data, and use the average values to make the
calculations which are described below. Between injections, wash the column with 10 mL of distilled water
at the same flow rate of 5—10 mL per minute. If colored material builds up on the column, repeat the
pretreatment procedure.
7. Calculate the resolution, selectivity, and efficiency.
Determine the following values. Show how each calculation is carried out and record your data in a table like
the one shown below. The shaded sections do not need to be filled in.
VR is retention volume. The retention volumes for the dyes in the experiment are the volumes corresponding
to the centers of the red and blue bands.
W is the band width, or the volume in mL of each dye as it emerges from the column.
VRavg is the total volume eluted at the center of the band of each of the dyes.
L is the column length. The cartridges in this experiment are 1.25 cm long.
The column radius is r. These cartridges have a radius of 0.5 cm.
VM is mobile phase volume. This represents about 50% of the total empty column volume and can be
estimated as VM = 0.5 it r2 L. The value of VM will be in cm3 (mL) if r and L are measured in centimeters.
k’ is the capacity factor. This is a unitless measure of the retention for each of the dyes, and can be calculated
as k’ = (VR - VM) / VM. The optimum range for k’ is between 1 and 10.
α is the selectivity or separation factor. It is the ratio of the separation of the k’ values: α = k’2 / k’1 where k’2
is the larger k’ value. For example, a value for a of 1.1 indicates that the column shows a 10% greater
rententivity for the component that elutes second. Generally, a mobile phase is chosen which gives a value
for a between 2 and 10.
N represents the number of theoretical plates in the column. This can be considered as the number of times a
solute is exchanged back and forth between the stationary and the mobile phase. The calculation is based on
the dye which is eluted last. Generally, columns with a larger value for N are more efficient. In the small
cartridges used, N should have a value between 20 and 200.
R is the resolution. This represents the major goal of the experiment, the measure of how well the two
components are separated by the column. R = (VRI - VR2) / ½ (W1 + W2). The numerator is the volume
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between bands. This is related to the selectivity. The denominator represents the average band width, which
is proportional to the efficiency of the column. As resolution increases above a value of 1, there is much
greater total separation of the dyes.
Data Table (Place your cursor in the white boxes and type your answers.)
Red Dye
Blue Dye
Experimental System
VR (start)
VR (end)
W = VR (end) - VR (start)
VRavg = VR (start) + ½ W
L
r
VM = 0.5 Π r2 L
k’ = (VRavg –VM)/VM
α = k’2 / k’1
N = 16 (VR/W)2
R = (VR1 –VR2) / ½ (W1 + W2)
Step Gradient Separation
In this type of procedure, the composition of the eluting liquid is changed. Since the column first a very polar
solvent, water, will be used. Then its composition will be changed to less polar more isopropanol. With this
procedure we will be able to separate the citric acid and flavoring oils the dyes.
8. Prepare the Isopropanol eluants.
Prepare the following concentrations of isopropanol in water by mixing the suggested amounts:
5% isopropanol in water: Mix 3.5 mL 70% isopropanol and 46.5 mL distilled water (or isopropanol and 47.2
mL distilled water).
28% isopropanol in water: Mix 20.0 mL 70% isopropanol and 30.0 mL distilled water
91% isopropanol and 34.5 mL distilled water).
9. Pretreat the cartridge.
Follow the same procedure as in step 3.
10. Inject the sample and elute the components.
Inject 1 mL of the grape drink. Elute the polar components of the mixture (citric acid and any sugar present)
by passing 5 mL of water through the column. Collect the effluent in a small beaker. Next, elute the red dye
by passing 5 to 10 mL of 6% isopropanol through the column. Note that large amounts of the 6%
isopropanol can be used without eluting the blue dye. Collect this effluent in a second beaker. Thirdly, use
the 28% isopropanol to elute the blue dye. Collect it in a third beaker. Lastly, use 8 mL of 70% isopropanol
to elute the polar flavor oils and other nonpolar additives. Collect this fraction in a fourth beaker.
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11. Evaporate the solvents and examine the components.
Allow the four beakers of solution to evaporate by leaving them in the fume hood until the next laboratory
period. Observe and describe the contents of each of the beakers.
Disposal
Solutions can be safely flushed down the sink. The cartridges can be discarded in the trash.
Discussion
In your laboratory report include answers to the following questions:
1. What is meant by polarity of molecules? What causes differences in polarity?
2. In discussing solubility, the rule “like dissolves like” is frequently used. What does this mean?
3. Draw the structural formula of isopropanol. Explain how it differs in polarity from water.
4. For good separation of the dyes, the resolution should be greater than one. What was the value you
calculated? Did the two dyes overlap as they emerged from the column, or was the separation a good one?
5. In the step gradient separation, four separate fractions were collected. How were these related to the
polarities of the column and of the eluting solvent?
6. Write a conclusion paragraph about what you learned and how to use this for science.